Glutathione quantification from protein-bound glutathione and erythrocyte lysate

The protocol utilized was the enzyme recycling assay described by Rahman et al. [23]. Total GSH, GSSG, and GSH was quantified from plasma protein-bound glutathione, erythrocyte lysate, and free plasma.

De-identified whole blood from each time point was immediately divided in two 2-mL portions in EDTA-treated tubes and placed at 4°C. One 2-mL portion was dedicated for analysis of GSH, GSSG, and total GSH from erythrocyte lysate and the other 2-mL portion was used to determine protein-bound glutathione. For analysis of protein-bound glutathione, the whole blood was spun at 1000g for 10 min at 4°C. The plasma supernatant was transferred and the remaining cell pellet was diluted with 2.6 mL of 0.1% Triton X-100 and 0.6% sulfosalicylic acid in KPE (0.1 M potassium phosphate buffer with 5 mM EDTA disodium salt, pH 7.5) for homogenization. To the viscous solution was added 1 mL of 1% NaBH₄ in KPE buffer for a 15-min reaction period at 22°C. The reaction was quenched using 0.4 mL 30% metaphosphoric acid in KPE, followed by centrifugation at 4°C, 1000g for 15 min. The supernatant was then used in the GSH, GSSG, and total GSH assays.

Sample preparation for erythrocyte lysate analysis commenced by centrifugation of the 2-mL portion at 4°C, 2500g for 5 min. The supernatant was removed and 4 mL of 5% metaphosphoric acid KPE solution was added for vigorous pellet re-suspension. The resultant solution was then spun at 3000g at 4°C for 10 minutes and the clear supernatant was collected for GSH, GSSG, and total GSH analysis.

The 96-well microplate reader was programmed to measure absorbance at 412 nm every 30 sec for 5 min. Reagent preparation was as follows: [5,5'-dithio-bis(2-nitrobenzoic acid)] (DTNB) was prepared as a 0.66 mg mL^–1 solution in KPE, 40 μL of a 250 units mL^–1 glutathione reductase (GR) stock was diluted with 3 mL KPE, and β-NADPH was also prepared as a 0.66 mg mL^–1 solution in KPE. GSH stock solutions were freshly prepared by dissolving 1 mg GSH mL^–1 in KPE. This GSH stock solution was diluted 1:100 with KPE to make a working solution of 10 μg mL^–1. Further dilution afforded a range of twofold concentrations from 0.103 nM to 26.4 nM. GSSG standards were prepared in a similar fashion to result in a 0.103–26.4 nM concentration series. Stock and sample solutions were kept at 4°C and protected from light during microplate loading.

For analysis of total GSH, the aforementioned erythrocyte lysate or plasma protein-bound glutathione solutions were added to the microplate wells in 20 μL volumes followed by immediate 120 μL addition of a freshly mixed solution of 1:1 GR and DTNB. Following a 30-sec incubation, 60 μL of β-NADPH was added and microplate reader data acquisition was performed. The analysis was performed in triplicate and repeated at least twice. Using the GSH and GSSG standards, the reaction rate (change in absorbance min^–1) was plotted versus a concentration range of 0.165 nM to 1.65 nM GSH or GSSG to construct calibration curves.

For analysis of GSSG, a 100 μL aliquot of erythrocyte lysate or plasma protein-bound glutathione solution was treated with 2 μL of 2-vinylpyridine as a 10% solution in KPE (v/v) at 22°C for 1 h in a well-ventilated fume hood. Following this incubation, 6 μL of triethanolamine was added as a 20% solution in KPE (v/v) for vigorous mixing. The solution was placed back at 4°C and microplate reader data was acquired as described above.

Intra- and inter-assay coefficient of variation (CV) values for erythrocyte lysate and protein-bound GSH data sets were calculated in-house to support repeatability of the enzymatic recycling assay described by Rahman et al [23]. Intra-assay CV values were calculated from data collected in triplicate and are found in the supplementary materials (S1 Table). Using the intra-assay CV values, the inter-assay CV values were 5.79 ± 3.35% and 5.89 ± 1.77% for erythrocyte lysate and protein-bound GSH, respectively.